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Abstract

β-amyloid (Aβ) and Tau proteins are biomarkers of Alzheimer's disease neuropathogenesis. We hypothesized that they are also potential biomarkers for postoperative cognitive dysfunction (POCD). The present study was designed to evaluate the use of the Aβ-42/Tau ratio for the diagnosis of POCD in patients undergoing hip/knee replacement surgery. A total of 80 patients who underwent total hip/knee replacement surgery were grouped into POCD or non-POCD patients at 7 days, 1 and 3 months after surgery, according to a z-score recommended by the international study of POCD. Cerebrospinal fluid samples were collected prior to surgery and the concentration of Aβ-42 and Tau protein were detected. The ratio of Aβ-42/Tau was compared between the two groups at different time points. The patients completed the whole battery of neuropsychological tests following surgery. The POCD occurrence rates at 7 days, 1 and 3 months were 40, 25 and 15%, respectively. The Aβ-42/Tau ratios were much lower in the POCD group than those in the non-POCD group at 7 days (1.7±0.4 vs. 2.6±0.3), 1 month (1.6±0.5 vs. 2.4±0.4), and 3 months (1.6±0.4 vs. 2.5±0.4). The sensitivity and specificity for diagnosis of POCD as indicated by the ratios, at the three time points, were 91.7 vs. 81.2%, 86.7 vs. 70%, and 88 vs. 75.5%, respectively. The results show the incidence of POCD of patients aged at least 65 years who have undergone hip or knee fracture surgery gradually decrease as more patients recover. However, the Aβ-42/Tau ratio can be used in forecasting the occurrence of POCD elderly patients.

Introduction

Postoperative cognitive dysfunction (POCD) is a
cognitive performance deterioration occurring mainly after
high-risk surgery (or anesthesia). It is characterized by
dysfunctions in mental activities, personality, interpersonal
communication and cognitive competence. Patients with POCD usually
complain of memory decline, and poor concentration and information
processing ability (1). POCD is
reversible in most patients, but a permanent cognitive impairment
can sometimes ensue. Prolonged POCD increases the risk of dementia,
and is associated with the development of Alzheimer's disease (AD)
(2,3). Therefore, it is of great importance to
diagnosis POCD as early as possible; nevertheless, the pathogenesis
of POCD is not clearly understood. It has been reported that the
patients' age, education level, intelligence and genetics, as well
as any underlying conditions, intraoperative factors and anesthesia
may all be risk factors for POCD (4). For example, elder patients, especially
those older than 65, are more susceptible to POCD than younger
patients (5). Animal studies have
shown that some anesthetic drugs impair the metabolism of acute
phase proteins, inducing β-amyloid proteins (Aβ) aggregation and
plaque deposition in rats, which significantly damage their memory
and spatial learning ability (6,7). During
surgery, reduced partial pressure of CO2 caused by
hyperventilation has also been reported to be associated with a
decrease of cerebral perfusion, thereby impairing the patient's
cognitive ability (8).

The frequency of POCD is also associated with
different invasive surgeries. Surgical invasion causes increased
levels of inflammatory cytokines in the peripheral and central
nervous system, which alter cognitive function by injuring the
hippocampus (9). Previously, an
increasing number of inflammatory cytokines have been recognized to
affect the hippocampus-related cognitive function (10,11).
Nevertheless, the neuropathogenesis remains unclear, and biomarkers
for the risk of POCD are not clearly determined.

Aβ, including 40 and 42, are components of AD senile
plaques. Tau is a microtubule-associated protein in neurons, which
is essential for microtubule formation and structural stability
(12). Both proteins have been
reported to play roles in AD neuropathogenesis, therefore, it is
possible that they may be effective biomarkers of POCD in
postoperative patients.

Patients undergoing hip-replacement surgery are
susceptible to POCD (11). In the
present study, we selected older patients, who underwent total
hip/knee replacement surgery, and detected the Aβ-42 and Tau levels
in cerebrospinal fluid (CSF) before and after surgery, to analyze
the diagnostic value of Aβ-42/Tau ratio on POCD occurrence. The
results of our study provide a theoretical basis for the clinical
diagnosis of POCD.

Materials and methods

Subjects

The Ethics Committee of People's Hospital of
Shouguang (Shandong, China) approved the study. Eligible patients
signed informed consent forms. A total of 80 patients (aged from 65
to 85 years) in American Standards Association (ASA), class II–III,
who received total hip arthroplasty (THR) or total knee replacement
(TKR) in our hospital, from October, 2014 to December, 2016 were
enrolled in the present study. Patients presenting with
preoperative mini-mental state examination (MMSE) scores <24,
with a history of neurological or psychiatric disease (Alzheimer,
stroke or psychological disease), with severe liver/renal
dysfunction, with serious visual or hearing disturbances, those
unable to communicate, speak or read, or were dependent on
psychoactive drugs or alcohol, were excluded from the study. The
WAIS (Wechsler Adult Intelligence Scale) (12) as well as the WBIS (Wechsler-Bellevue
scale) (13) were used for
neuropsychology tests before and after surgery. The incidence of
POCD was evaluated by the z-score method. Patients were classified
into POCD and non-POCD groups according to the cognitive condition.
A group of 80 healthy volunteers aged 65–85 years were recruited in
our study as the control group.

Anesthesia management

The patients received standard anesthesia. Once in
the operating room, the arterial blood pressure, electrocardiogram
and SPO2 (saturation of pulse oximetry) were monitored.
The patients were placed in a lateral position for combined
spinal-epidural anesthesia. CSF was collected from each patient
after subarachnoid puncture and stored at −80°C for subsequent
protein measurements. Anesthesia was induced with 0.75% ropivacaine
hydrochloride (2 ml; AstraZeneca AB, Sodertalje, Sweden) in a 10%
glucose injection (0.5 ml)+CSF (0.5 ml). After catheterization with
a 4 cm epidural guiding tube, the patients were provided oxygen
inspiration through mask (2 l/min). The systolic pressure was
controlled and maintained >100 mmHg (5–10 mg ephedrine; Hong Hui
Pharmaceutical Co., Ltd., Zhengzhou, China) were injected
intravenously if the blood pressure decreased >30% of the basal
value). The patients underwent THR or TKR during anesthesia and
conventional perioperative care with analgesia as needed.

Neuropsychology test

The patients underwent neuropsychological tests
prior to surgery, and then 7 days, 1 and 3 months after surgery.
The test items included several different areas. To evaluate
summation the subjects were required to continuously add the same
number (3 or 4) from 1 to 49. The time spent in total, the errors
and missing numbers were recorded to evaluate the subject's
attention. The evaluation of visual reproduction included a visual
learning and memory test requiring the subjects to recall and draw
3 images they were shown. The digit span is a test requiring
subjects to repeat a group of digits forward, and then backwards.
To test language memory the subjects were given an associative
learning test where they had to remember specifid terms they had
heard in the allotted time. To evaluate processing speed, the
subjects were given the digit symbol test, where they had to match
numbers from 1 to 9 according to a given key within 90 sec. The
last cognitive function test given was the ligature test, where the
subjects connected a group of digits in order, and the time spent
on the task was recorded.

Neuropsychology experts who had received
professional training in a special room and at the same time
performed all the above tests. Healthy individuals in the control
group underwent the same cognitive assessments at the same time
intervals as matching subjects in the patient's group.

Diagnosis of POCD

A z-score recommended by the International Study of
Postoperative Cognitive Dysfunction (ISPOCD) was calculated for the
diagnosis of POCD. First, the differences between the post- and
pre-operative scores were calculated, then the test scores were
divided by the standard deviation. If the z-score was >1.96 for
>2 times, the patient was classified as manifesting POCD
(5,14).

Concentration of Aβ-42 and Tau
proteins

The levels of Aβ-42 and Tau proteins in CSF were
measured using ELISA kits (Nanjing Jiancheng Bioengineering
Research Institute, Nanjing, China) according to the manufacturer's
instructions. Briefly, samples were assayed in triplicate (sample,
standard, blank control) on ELISA plates coated with human
anti-amyloid β (anti-Aβ1-42) or anti-Tau antibodies, and then
positive samples were detected by horseradish peroxidase. The
products were stained using tetramethyl benzidine for 15 min. The
reactions were analyzed on the microplate reader (RT-2,100 C;
Shenzhen Keeptop Electronic Co., Ltd., Shenzhen, China) at 450
nm.

Statistical analysis

Data were analyzed using SPSS 17.0 software (SPSS
Inc. Chicago, IL, USA) software. Data were expressed as means ± SD
or percentage, and analyzed by t-test or χ2 test,
respectively. The correlation between the Aβ-42/Tau proteins and
z-scores were analyzed using the Pearson's correlation analysis.
The cut-off value was determined by ROC (receiver operator
characteristic) working curve. P<0.05 was considered to indicate
a statistically significant difference.

Results

General data between patients and
normal controls, and between POCD and non-POCD

All 80 patients were successfully followed up, and
the neuropsychological tests were completed on the 80 normal
controls.

The percentages of patients experiencing POCD were
40 (32/80), 25 (20/80) and 15% (12/80) at 7 days, 1 and 3 months
postoperatively, respectively. The details of general parameters
for patients and normal controls are listed in Table I. There were no significant
differences in terms of age, weight, height, sex, or years of
education between the normal controls and the patients.

Table I.

General characteristics of
patients.

Table I.

General characteristics of
patients.

Items

Normal control
(n=80)

Patients (n=80)

Age (mean ± SD,
years)

73.6±5.8

74.5±4.8

Weight (mean ± SD,
kg)

57.2±5.1

58.5±4.9

Height (mean ± SD,
cm)

162.1±7.9

163.2±8.0

ASA classification
(II/III)

22/58

42/38

Years of education
(years)

9±3

10±2

[i] There are no
significant differences between the two groups.

Neuropsychology test results on the
7th day after surgery

On the 7th day after surgery, there were 32 patients
with POCD. For general data between patients presenting POCD (n=32)
and those not affected by it (n=48), no significant differences
were found in terms of anesthesia time, operation duration,
bleeding volume during surgery or amount of ephedrine used
(Table II).

Table II.

General characteristics of patients in
normal and patients groups (POCD and non-POCD group) on the 7th day
post-operation.

Table II.

General characteristics of patients in
normal and patients groups (POCD and non-POCD group) on the 7th day
post-operation.

Items

POCD (n=32)

Non-POCD (n=48)

Age (mean ± SD,
ages)

74.3±5.2

74.3±4.6

Weight (mean ± SD,
kg)

59.9±5.1

57.5±4.6

Height (mean ± SD,
cm)

161.7±8.0

162.5±8.2

ASA classification
(II/III)

10/22

15/33

Years of education
(mean ± SD)

7±1.6

8±2.3

Anesthesia time (mean
± SD, min)

132±22

135±24

Time of operation
(mean ± SD, min)

93±16

90±14

Bleeding volume
during surgery (mean ± SD, ml)

207±94

205±92

Ephedrine use
(%)

58

60

[i] There were no
significant differences between the two groups; POCD, postoperative
cognitive dysfunction; ASA, American Standards Association.

Neuropsychology test results on the
1st month after surgery

One month after surgery, the number of patients
suffering from POCD was reduced to 20. The general data between
those affected and those not affected by POCD were not
significantly different (Table
IV).

Table IV.

General characteristics of patients
in normal control and patients group (POCD group and non-POCD) at 1
month post-operation.

Table IV.

General characteristics of patients
in normal control and patients group (POCD group and non-POCD) at 1
month post-operation.

Items

POCD (n=20)

Non-POCD
(n=60)

Ages (mean ± SD,
ages)

75.1±5.2

74.6±5.1

Weight (mean ± SD,
kg)

60.2±5.1

59.4±4.5

Height (mean ± SD,
cm)

162.3±8.4

161.8±8.9

ASA classification
(II/III)

8/12

16/44

Years of education
(mean ± SD)

8±2

8±2

Anesthesia time
(mean ± SD, min)

130±22

132±24

Time of operation
(mean ± SD, min)

95±16

93±15

Bleeding volume
during surgery (mean ± SD, ml)

205±94

205±92

Ephedrine use
(%)

56

60

[i] There were no
significant differences between the two groups; POCD, postoperative
cognitive dysfunction; ASA, American Standards Association.

Neuropsychology test results 3 months
after surgery

Three months after surgery, 12 patients were
reported to suffer from POCD. There were no significant differences
in basic clinical data between those affected by POCD and those who
were not affected (Table VI).

Table VI.

General characteristics of patients
in the normal control and patients group (POCD and non-POCD group)
at 3 months post-operation.

Table VI.

General characteristics of patients
in the normal control and patients group (POCD and non-POCD group)
at 3 months post-operation.

Items

POCD (n=20)

Non-POCD
(n=60)

Age (mean ±
SD)

75.7±5.2

75.3±4.9

Weight (mean ± SD,
kg)

61.8±5.2

60.3±5.4

Height (mean ± SD,
cm)

163.3±8.1

162.1±8.7

ASA classification
(II/III)

4/8

22/46

Years of education
(mean ± SD)

8±2

8±2

Anesthesia time
(mean ± SD, min)

135±24

132±22

Time of operation
(mean ± SD, min)

97±16

95±16

Bleeding volume
during surgery (mean ± SD, ml)

210±95

207±92

Ephedrine use
(%)

57

59

[i] There were no
significant differences between the two groups; POCD, postoperative
cognitive dysfunction; ASA, American Standards Association.

Concentrations of Aβ-42 and Tau
proteins after surgery

The concentration of Aβ-42 and Tau proteins in the
CSF samples were detected to evaluate nervous system injury after
surgery. The results showed that at 7 days, 1 and 3 months after
surgery, the Aβ-42 proteins were significantly reduced in the
samples of those affected by POCD (Fig.
1). However, the Tau proteins did not seem to be affected, and
there were no significant differences between the two groups of
patients.

The ratios of Aβ-42 and Tau in patients affected by
POCD were much lower than those of unaffected patients (P<0.05),
and it they negatively correlated with the z-scores (r, −0.681,
P< 0.05; r, −0.643, P<0.05; r, −0.620, P<0.05,
respectively for post-operative 7 days, 1 and 3 months). The
sensitivity and specificity of the ratios for POCD occurrence were
91.7 and 81.2, 86.7 and 70.0, 88.0 and 75.5% for 7 days, 1 month,
and 3 months, respectively, after surgery time points.

Discussion

The pathogenesis of POCD is currently unclear. Thus,
the identification of biomarkers for POCD has drawn much attention
by neurologists. Previous studies have confirmed the diagnostic
value of Aβ and Tau proteins for AD (15,16). It
is thought that POCD shares similar neuropathological mechanisms
with AD (17). Thus, Aβ and Tau
proteins are hypothesized to be similarly useful for the diagnosis
of POCD.

Several recent studies have evaluated the diagnostic
efficacy of CSF Aβ and Tau proteins for POCD after different
procedures. For example, Evered et al found that protein
levels of Aβ42 and 40 in the blood are associated with the
cognitive dysfunction after cardiac surgery (18). Two Chinese studies have also
suggested that the patients undergoing hip/knee replacements with
low CSF Aβ42 levels had high risk of cognitive dysfunction
(11,19). However, the authors of the two
studies did not use the POCD diagnostic criteria recommended by the
International Society of Preventive and Community Dentistry (ISPCD)
group. Until recently, the ISPCD diagnostic criteria for POCD had
not been used extensively in China. Our study evaluated the
correlation between the CSF Aβ42/Tau ratio and the scores of
international cognitive tests. The results show that the percentage
of patients who develop POCD after hip/knee replacement were 40, 25
and 15% at 7 days, 1 and 3 months postoperatively, respectively.
Additionally, in agreement with the findings from previous studies,
our study has also demonstrated the diagnostic effect of the
Aβ42/Tau ratio on POCD patients.

The evaluation of the patient's neuropsychology is
essential for the diagnosis of POCD. There are many criteria to
take into account for detection of neuropsychological
abnormalities. The ISPOCD recently recommended a battery of
neuropsychology tests for assessing patient's mental states. This
includes several tests such as the stroop color-word, the visual
language learning and the alphabet number coding test. The WAIS and
WBIS tests used for this study are also included in the ISPOCD
recommendations. They are applicable to Chinese patients for
evaluation of cognitive function with high sensitivity and
specificity (20). However, the test
methods are time-consuming, and therefore may not be well tolerated
by patients. To prevent problems in this study, we used the MMSE
preoperatively for preliminary screening.

It has been suggested that an inflammatory response
may be associated with decline of neurocognition after anesthesia
or surgery (9,21,22).
Previous studies have also shown that both anesthesia and surgery
are associated with the Aβ and Tau proteins in CSF (23,24). A
central cholinergic system of neurotransmitters can regulate
memory, consciousness and learning of an individual (25). As members of ligand-gated cation
channels, nicotinic acetylcholine receptors play key roles in the
neurotransmitter system. In this context, the Aβ proteins are
reported to have adverse effects on the synthesis and release of
acetylcholine (26). It has been
previously shown that overexpression of Aβ proteins profoundly
damages an individual's learning memory and hippocampal volume
(27).

The neuropathological characteristics for AD include
the presence of extracellular plaques of Aβ peptides and
neurofibrillary tangles formed by hyperphosphorylated Tau proteins
(16,28). Tau is a microtubule-associated
protein and has 6 isoforms in humans. In pathological conditions
such as craniocerebral injury, Tau proteins assemble and are
hyperphosphorylated to form neurofibrillary tangles (29,30).
Reports have shown that under anesthesia of any kind a transient
and reversible high phosphorylation of Tau protein is induced.
However, Tau phosphorylation is probably associated with the lower
temperature in the body caused by the anesthesia (31) and the content of Tau in CSF is not
affected.

In this study, we chose the ratio of Aβ-42/Tau for
evaluation of POCD risk for two reasons. First, Aβ-42 proteins are
more liable to aggregate and deposit outside of cells, causing much
more toxicity to neurons than Aβ-40, and second, previous studies
have demonstrated that the Aβ-42/Tau ratio is an important
biomarker in the diagnosis of AD. Moreover, the Aβ-42/Tau ratio is
more sensitive for AD detection than Aβ-42 or Tau alone. Our
results showed that there were significant differences in the
ratios between the patients affected by POCD and those not affected
at 7 days (sensitivity, 91.7 and specificity, 81.2%), one month
(sensitivity, 6.7 and specificity, 81.2%) and 3 months
(sensitivity, 86.7 and specificity, 70%) after surgery. These
results indicate that the ratio of Aβ-42/Tau can be used to predict
the occurrence of POCD.

In conclusion, there are significant associations
between the preoperative and postoperative Aβ-42/Tau ratio and
cognition dysfunctions. The results of this study agree with other
reported studies where the Aβ-42/Tau ratio is a potential biomarker
for diagnosing POCD.